Esters of 7-sulfenyliminocephem-4-carboxylic acids

ABSTRACT

Alkoxylated β-lactam compound useful as an intermediate for various cephalosporin (or penicillin) derivatives which may be prepared by reacting a 7-sulfenylaminocephem compound (or a 6-sulfenylaminopenam compound) with an oxidizing agent or reacting a sulfinylaminocephem compound (or a 6-sulfinylaminopenam compound) with a dehydrating agent to give a sulfenyliminocephem compound (or a 6-sulfenyliminopenam compound) and reacting the latter compound with an alcohol in the presence of a base, an organic acid or a Lewis acid.

This is a continuation of application Ser. No. 907,772 filed May 19,1978, abandoned, which, in turn, is a division of U.S. Ser. No. 779,907,filed Mar. 21, 1977 (now abandoned).

This invention relates to new alkoxylated β-lactam compounds and a newprocess for the preparation thereof.

More particularly, it relates to new alkoxylated β-lactam compoundshaving the formula ##STR1## (wherein R¹ represents a trihalogenomethylgroup or an aromatic hydrocarbyl group optionally substituted with fromone to 5 members selected from nitro, cyano, halogen or alkoxycarbonyl,R² represents an alkyl group, R³ represents carboxyl group or aprotected carboxyl group and Z represents a fragment of the formula##STR2## (wherein R⁴ represents alkyl, alkoxy, halogen,carbamoyloxymethyl, acyloxymethyl, 5- or 6-membered heterocyclicthiomethyl optionally having substituted or unsubstituted alkyl) and apharmaceutically acceptable salt thereof.

In the above formula (I),

R¹ may be a trihalogenomethyl group, for example, trichloro-, tribromo-or trifluoro methyl; or an aromatic hydrocarbyl group optionallysubstituted with from one to 5 members selected nitro, cyano, halogen oralkoxycarbonyl, for example, phenyl, o-(m- or p-) nitrophenyl, o-(m- orp-)chlorophenyl, o-(m- or p-)bromophenyl, o-(m- or p-)cyanophenyl, o-(m-or p-)methoxycarbonylphenyl, 2,4-dinitrophenyl, 2,4-dichlorophenyl,2,4,6-trichlorophenyl, pentachlorophenyl, and naphthyl, preferablypentachlorophenyl, pentabromophenyl and mono- or dinitrophenyl, e.g., o-or p-nitrophenyl, and 2,4-dinitrophenyl.

R² is preferably an alkyl having 1-4 carbon atoms, for example, methyl,ethyl, n-propyl, isopropyl and n-butyl.

R³ is carboxyl group or a protected carboxyl group such as analkoxycarbonyl group having 1-4 carbon atoms in the alkyl moiety, e.g.,methoxycarbonyl, ethoxycarbonyl, n-propoxycarbonyl, isopropoxycarbonyl,n-butoxycarbonyl, isobutoxycarbonyl, t-butoxycarbonyl, ahalogenoalkoxycarbonyl group having 1-4 carbon atoms in the alkylmoiety, e.g., dichloroethoxycarbonyl or trichloroethoxycarbonyl, abenzyloxycarbonyl group optionally substituted with halogen, methoxy ornitro, e.g., benzyloxycarbonyl, p-chlorobenzyloxycarbonyl,p-methoxybenzyloxycarbonyl or p-nitrobenzyloxycarbonyl,diphenylmethyloxycarbonyl group, a trialkylsilyloxycarbonyl group having1-4 carbon atoms in each alkyl moiety, e.g., trimethylsilyloxycarbonylor triethylsilyloxycarbonyl, a dialkylhalogenosilyloxycarbonyl grouphaving 1-4 carbon atoms in each alkyl moiety, e.g.,dimethylchlorosilyloxycarbonyl or dimethylbromosilyloxycarbonyl, aphenacyloxycarbonyl group optionally substituted with halogen, ormethoxy, e.g., p-chlorophenacyloxycarbonyl, p-bromophenacyloxycarbonyl,p-methoxyphenacyloxycarbonyl or an acyloxycarbonyl, e.g.,acetoxycarbonyl or benzoyloxycarbonyl, a halogenoacylcarbonyl group,e.g., chloroacetoxycarbonyl or bromoacetoxycarbonyl adihalogenophosphinooxycarbonyl group, e.g., dichlorophosphinooxycarbonylor dibromophosphinooxycarbonyl, a dialkylphosphinooxycarbonyl group,e.g., dimethylphosphinoxycarbonyl or an iminocarbonyl group, e.g.,3-oxo-2,3-dihydro-s-triazolo(4,3-a)pyridone-3-ylcarbonyl orsaccharylcarbonyl.

R³ is preferably t-butoxycarbonyl, diphenylmethyloxycarbonyl and aphenacyloxycarbonyl group optionally substituted with chlorine orbromine.

R⁴ is preferably an alkyl having 1-4 carbon atoms, alkoxy having 1-4carbon atoms, halogen, carbamoyloxymethyl, alkanoyloxymethyl having 1-4carbon atoms in the alkyl moiety, benzoyloxymethyl or heterocyclicthiomethyl selected from the group consisting of tetrazolyl,1-alkyltetrazolyl-, 1-carboxyalkyltetrazolyl-,1-alkoxycarbonylalkyltetrazolyl-, 1-sulfoalkyltetrazolyl-,1-aminosulfonylalkyl-, 1-mono- or dialkylaminosulfonylalkyltetrazolyl-,1-aminoalkyltetrazolyl-, 1-mono- or dialkylaminoalkyltetrazolyl-,isoxazolyl-, imidazolyl-, thiazolyl-, triazolyl-, thienyl-,thiadiazolyl-, methylthiadiazolyl-, pyrimidinyl- and pyridylthiomethyl.

As the pharmaceutically acceptable salts, there may be mentioned saltsof alkali or alkaline earth metals, e.g., sodium, potassium, calcium,aluminum salt, ammonium salt, or salts of organic bases, e.g.,triethylamine, dicyclohexylamine, dibenzylamine, dimethylbenzylamine,piperidine or N-ethylpiperidine.

Recently, there have been isolated 7α-methoxycephalosporins from acultured broth of bacteria belonging to genus Streptomyces (The Journalof the American Chemical Society, 93, 2308; Antimicrobial Agents andChemotherapy, 2, 122; Japanese Provisional Patent Publication Nos.26488/74, 30593/74, 42893/74) and various methods for introducing analkoxy group into the 7-position of cephem nucleus have been developed.These methods may be classified as follows: (a) a method in which aSchiff base of a 7-aminocephem compound is treated with a strong base toproduce a carbanion at the 7-position and then the carbanion is directlyalkoxylated, or said Schiff base is once halogenated or alkylthiolatedand then one of these electrophilic groups is converted into an alkoxygroup (Japanese Provisional Patent Publication No. 42691/72, TetrahedronLetters, 273 and 3505 (1973); Journal of the Organic Chemistry, 38, 943and 2857 (1973); Journal of the Organic Chemistry, 39, 2794 (1974); (b)a method in which an alkoxy group is introduced after diazotization of a7-aminocephem compound (The Journal of the American Chemical Society,94, 1408 (1972); (c) a method in which a 7-acylaminocephem compound isconverted after N-chlorination thereof, into a compound having anacylimino group and an alcohol is added thereto (The Journal of theAmerican Chemical Society, 95, 2403 (1973); Tetrahedron Letters, 1311(1974); Journal of the Organic Chemistry, 38, 1436 (1973); (d) a methodin which 7-benzylideneaminocephem compound is oxidized followed byreacting an alcohol (German Patent Laid Open No. 2442540); and (e) amethod in which 7β-(α'-halogeno)cephem compound is reacted with ahalogenating agent to give a α-halogenoimminohalide, the product isreacted with an alkali metal alkoxide followed by hydration (GermanPatent Laid Open No. 2512670). Each of these methods, however, hasseveral deffects. Namely, in method (a), the direct alkoxylation ofcarbanion at the 7-position of cephem compound gives poor yield and theindirect method, in which an electrophilic group is once introduced andthen the group is converted into an alkoxy group, increases the numberof steps in the process. In method (b), many steps for reaction isinvolved, the procedures are complicated and the yield is not good. Inmethod (c), when a anion-forming position exists on a side chain at the7- or 3-position, the object can not be achieved. In method (d), the7-alkoxylated-7-benzylideneaminocephem compound obtained can not bedirectly converted to 7-alkoxylated-7-acylaminocephem compound. Inmethod (e), a certain kind of the starting 7β-(α-halogeno)cephemcompound is difficult to be synsesized.

As a result of our earnest studies on alkoxylation of a cephem nucleusat its 7-position or a penam nucleus at its 6-position, we have foundand developed an improved method which entirely differs from the priormethods and can be applied to various β-lactam compounds.

It is thus an object of the present invention to provide an alkoxylatedβ-lactam compound useful as an intermediate for the synthesis of various7-alkoxylated cephalosporin or 6-alkoxylated penicillin derivatives eachhaving a broad antibacterial spectrum.

It is another object of the present invention to provide a process forthe preparation of such an alkoxylated β-lactam compound. Other objectsand advantages of this invention will become apparent from the followingdisclosure of this invention.

According to the present invention, the alkoxylated β-lactam compoundhaving the formula (I) can be prepared by reacting a sulfenylaminoβ-lactam compound having the formula ##STR3## (wherein R¹, R³ and Z havethe same meanings as defined above) with an oxidizing agent; or reactinga sulfinylamino β-lactam compound having the formula ##STR4## (whereinR¹, R³ and Z have the same meanings as defined above) with a dehydratingagent to give a sulfenylimino β-lactam compound having the formula##STR5## (wherein R¹, R³ and Z have the same meanings as defined above)and reacting the latter compound (III) with an alcohol having theformula

    R.sup.2 OH                                                 (IV)

(wherein R² has the same meaning as defined above) in the presence of abase, an organic acid or a Lewis acid.

In carrying out the process of this invention, the compound (III) isprepared by reacting the compound (II) with an oxidizing agent. Theoxidizing agent in this reaction includes a metal oxide, such asmanganese dioxide, potassium permanganate, chromic anhydride, potassiumdichromate; lead tetraacetate; potassium nitrosodisulfonate,2,3-dichloro-5,6-dicyanobenzoquinone; tetrachloro-1,4-benzoquinone;N-chlorosuccinimide; trichloroisocyanuric acid; and sulfuryl chloride.Manganese dioxide can be most preferably employed. The reaction may bepreferably carried out in an inert organic solvent under stirring at atemperature of from -100° C. to 100° C. for 30 minutes-3 hours. Suitablesolvent is an aprotic solvent, for example, benzene, chloroform,methylene chloride, acetonitrile, dioxane, ethyl acetate. In case whereN-chlorosuccinimide, trichloroisocyanuric acid or sulfuryl chloride isused as the oxidizing agent, it is desirable to carry out the reactionin the presence of a tertiary amine, for example, quinoline,diethylaniline, dimethylaniline, pyridine, triethylamine, trimethylamineand diazabicyclooctane. After completion of the reaction, the desiredproduct can be isolated and purified by conventional means.

Alternatively, the compound (III) can be prepared by reacting thecompound (II)' with a dehydrating agent in the presence of a base. Thedehydrating agent employed in this reaction includes thionyl halides,e.g., thionyl chloride and thionyl bromide; carboxylic acid anhydridesor halides, e.g., acetic anhydride, trifluoroacetic anhydride, acetylchloride and trifluoroacetyl chloride; organic sulfonic acid anhydridesor halides, e.g., benzenesulfonic anhydride, p-toluenesulfonicanhydride, benzenesulfonyl chloride, p-toluenesulfonyl chloride;phosgene; and phosphrus oxychloride. Thionyl chloride may be mostpreferably used. The reaction is preferably carried out in an aproticorganic solvent under stirring at a temperature of from -100° C. to aroom temperature for 5 minutes-12 hours. Suitable solvent is chloroform,methylene chloride, tetrahydrofuran, ether and ethyl acetate. The baseincludes a tertiary amine, for example, quinoline, diethylaniline,dimethylaniline, pyridine, triethylamine, trimethylamine anddiazabicyclooctane; and an alkali metal carbonate or bicarbonate, forexample, sodium carbonate, sodium bicarbonate, potassium carbonate,potassium bicarbonate. After completion of the reaction, the desiredproduct can be isolated and purified by conventional means.

The reaction mixture including the compound (III) can be employedwithout isolation as a starting material in the next step.

The compound (I) is prepared by reacting the compound (III) with thealcohol (IV) in the presence of a base, an organic acid or a Lewis acid.The base employed in this reaction includes an organic base such as atertiary amine, for example, quinoline, diethylaniline, dimethylaniline,pyridine, triethylamine, trimethylamine and diazabicyclooctane; and aninorganic base such as an alkali or alkaline earth metal hydroxide, forexample, sodium hydroxide, potassium hydroxide, calcium hydroxide; analkali metal carbonate, for example, sodium carbonate and potassiumcarbonate, and an alkali metal alkoxide, for example, sodium methoxide,lithium methoxide, potassium methoxide, potassium t-butoxide, sodiumt-butoxide. There may be most preferably used an alkali alkoxide havingthe formula R² OM (wherein R² has the same meaning as defined above andM is alkali metal). The organic acid includes methanesulfonic acid,benzenesulfonic acid and p-toluenesulfonic acid and the Lewis acidincludes zinc chloride, borontrifluoride etherate.

The reaction may be preferably carried out in an inert organic solventat a temperature of from -100° C. to 50° C., preferably -40° C. to -20°C., for 5 minutes-10 hours. After completion of the reaction, thedesired product can be isolated and purified by conventional means.

The compound (II) and (II)', which are used as a starting material inthis invention, can be prepared by reacting a compound having theformula ##STR6## (wherein R³ and Z have the same meanings as definedabove) with a sulfenyl halide having the formula

    R.sup.1 --S--X.sup.1

(wherein R¹ has the same meaning as defined above and X¹ represents ahalogen atoms, e.g., chlorine and bromine) or a sulfinyl halide havingthe formula ##STR7## (wherein R¹ has the same meaning as defined aboveand X² represents a halogen atom, e.g., chlorine and bromine)respectively, in the presence of a hydrogen halide acceptor such as abase or an oxirane. The base includes a tertiary amine, e.g.,triethylamine, dimethylamine, pyridine; an alkali metal hydroxide, e.g.,sodium hydroxide, potassium hydroxide; and an alkali metal carbonate orbicarbonate, e.g., sodium carbonate, sodium bicarbonate, potassiumcarbonate and potassium bicarbonate. The oxirane encludes ethylene oxideand propylene oxide. The reaction is preferably carried out in an inertorganic solvent, e.g., chloroform, methylene chloride, dioxane,tetrahydrofuran, at a temperature from -100° C. to a room temperature.

The compound (I) obtained in this invention can be converted to variousknown 7β-acylamino-7α-alkoxycephalosporins or6β-acylamino-6α-alkoxypenicillins which have excellent antibacterialactivities by reaction with organic carboxylic acid halides in an inertsolvent. Alternatively, the compound (I) can be converted to7β-amino-7α-alkoxycephalosporins or 6β-amino-6α-alkoxypenicillins whichare useful as intermediates for β-lactam antibiotics by reaction with anucleophile such as sodium iodide, thiourea, thiophenol, sodium azide,thioglycolic acid, potassium thiocyanate and sodium thiosulfate,hexamethylphosphorous triamide. The following examples and referentialexamples are given for purpose of illustrating of this invention. It isto be understood that these examples and referential examples should notbe construed as limiting the scope of this invention.

Examples 1 to 4 illustrate the preparation of the compound (I) from thecompound (III).

Examples 5 to 14 illustrate the preparation of the compound (I) from thecompound (II) or (II)' via the compound (III).

Referential examples 1 to 5 illustrate the preparation of the compound(II) and (II)' from the compound (V).

Referential examples 6 to 9 illustrate the preparation of the compound(VI) from the compound (I).

Referential examples 4 to 6 illustrate the preparation of the compound(VII) from the compound (I).

Reaction sequence in this invention may be schematically shown asfollows: ##STR8## R¹, R², R³, Z, X¹ and X² have the same meanings asdefined above and R represents an acyl group.

EXAMPLE 17α-Methoxy-3-methyl-7β-orthonitrophenylsulfenylamino-3-cephem-4-carboxylicacid tert-butyl ester

In 60 ml. of anhydrous methanol was suspended 1.72 g. of3-methyl-7-orthonitrophenylsulfenylimino-3-cephem-4-carboxylic acidtert-butyl ester. To the suspension was added under stirring at -78° C.a solution of lithium methoxide which was prepared from 170 mg. (24.5mmol.) of metallic lithium and 24 ml. of anhydrous methanol. Afterstirring at -78° C. for 30 minutes, 70 ml. of anhydrous tetrahydrofuranwas gradually added to the mixture and the reaction mixture was furtherstirred for 3.5 hours at -78° C. to produce a transparent solution.After 2.0 ml. of acetic acid was added to complete the reaction and then50 ml. of water was added to the reaction mixture, the thus obtainedmixture was extracted three times with 100 ml. of ethyl acetate. Afterwashing with water and drying, the solvent was removed by evaporationunder reduced pressure to obtain a crude product. The crude product wassubmitted to purification by column-chromatography in which silica gelwas used as a packing and a mixture of benzene-ethyl acetate (10:1) wasused as an eluent to isolate 1.17 g. of7α-methoxy-3-methyl-7β-orthonitrophenylsulfenylamino-3-cephem-4-carboxylicacid tert-butyl ester melting at 149°-150° C.

Infrared Spectrum (Nujol): 3300, 1765 cm⁻¹

Nuclear Magnetic Resonance Spectrum (CDCl₃) δppm: 1.50 (9H, singlet),2.12 (3H, singlet) 3.18 and 3.39 (2H, AB-quartet, J=18 Hz) 3.57 (3H,singlet), 4.30 (1H, singlet) 4.90 (1H, singlet), 7.12-8.35 (4H,multiplet)

EXAMPLE 27α-Methoxy-3-methyl-7β-(2',4'-dinitrophenyl)sulfenylamino-3-cephem-4-carboxylicacid tert-butyl ester

In 30 ml. of anhydrous methanol was suspended 1.35 g. of3-methyl-7-(2',4'-dinitrophenyl)sulfenylimino-3-cephem-4-carboxylic acidtert-butyl ester. To the suspension was added under stirring at -78° C.a solution of lithium methoxide which was prepared from 102 mg. ofmetallic lithium and 12 ml. of anhydrous methanol. After stirring at-78° C. for 30 minutes, 35 ml. of anhydrous tetrahydrofuran wasgradually added to the mixture and the reaction mixture was furtherstirred for 3.5 hours at -78° C. to produce a transparent solution.After 1.0 ml. of acetic acid was added to complete the reaction and then25 ml. of water was added to the reaction mixture, the thus obtainedmixture was extracted three times with 50 ml. of ethyl acetate. Afterwashing with water and drying, the solvent was removed by evaporationunder reduced pressure to obtain a crude product. The crude product wassubmitted to purification by columnchromatography in which silica gelwas used as a packing and a mixture of benzene-ethyl acetate (10:1) wasused as an eluent to isolate 1.20 g. of7α-methoxy-3-methyl-7β-(2',4'-dinitrophenyl)sulfenylamino-3-cephem-4-carboxylicacid tert-butyl ester.

Infrared Spectrum (Nujol): 3300, 1780 cm⁻¹

Nuclear Magnetic Resonance Spectrum (CDCl₃) δppm: 1.49 (9H, singlet),2.12 (3H, singlet) 3.20 and 3.40 (2H, AB-quartet, J=18 Hz) 3.58 (3H,singlet), 4.58 (1H, singlet) 4.93 (1H, singlet), 8.35-9.08 (3H,multiplet)

EXAMPLE 37α-Methoxy-3-acetoxymethyl-7β-orthonitrophenylsulfenylamino-3-cephem-4-carboxylicacid benzhydryl ester

In a mixture of 60 ml. of methanol and 40 ml. of benzene was dissolved515 mg. of3-acetoxymethyl-7-orthonitrophenylsulfenylimino-3-cephem-4-carboxylicacid benzhydryl ester. To the solution was added with stirring at roomtemperature 155 mg. of paratoluenesulfonic acid hydrate to effectreaction. After 30 minutes, a saturated solution of sodium bicarbonatewas added to stop the reaction and the reaction mixture was extractedthree times with ethyl acetate. After washing with water and drying, thesolvent was removed by evaporation under reduced pressure to obtain acrude product. The crude product was submitted to purification bycolumn-chromatography in which a mixture of benzene-ethyl acetate (10:1)was used as an eluent and silica gel was used as a packing to isolate388 mg. of7α-methoxy-3-acetoxymethyl-7β-orthonitrophenylsulfenylamino-3-cephem-4-carboxylicacid benzhydryl ester.

EXAMPLE 4 6α-Methoxy-6β-orthonitrophenylsulfenylaminopenicillanic acidparabromophenacyl ester

In 15 ml. of anhydrous methanol was suspended 558 mg. of6-orthonitrophenylsulfenyliminopenicillanic acid parabromophenacylester. To the suspension was added under stirring at -78° C. a solutionof lithium methoxide which was prepared from 36.2 mg. of metalliclithium and 6 ml of anhydrous methanol. After stirring at -78° l C. for30 minutes, 18 ml. of anhydrous tetrahydrofuran was gradually added tothe mixture and the reaction mixture was further stirred for 3.5 hoursat -78° C. to produce a transparent solution. After 0.5 ml. of aceticacid was added to complete the reaction and then 12 ml. of water wasadded to the reaction mixture, the thus obtained mixture was extractedthree times with 25 ml. of ethyl acetate. After washing with water anddrying, the solvent was removed by evaporation under reduced pressure toobtain a crude product. Subsequently, the crude product was submitted topurification by preparative silica gel thin-layer-chromatography(thickness: 0.2 cm., 20×40 cm. development system: benzene-ethyl acetate(10:1)) and a component having a Rf value of 0.4 was isolated to obtain182 mg. of 6α-methoxy-6β-orthonitrophenylsulfenylaminopenicillanic acidparabromophenacyl ester melting at 148°-150° C.

Infrared Spectrum (Nujol): 3300, 1790 cm⁻¹

Nuclear Magnetic Resonance Spectrum (CDCl₃) δppm: 1.68 (6H, singlet),3.53 (3H, singlet) 4.30 (1H, singlet), 4.80 (1H, singlet) 5.38 (2H,singlet), 5.45 (1H, singlet) 7.23-8.38 (8H, multiplet)

EXAMPLE 5 6α-Methoxy-6β-orthonitrophenylsulfenylaminopenicillanic acidparabromophenacyl ester

In 100 ml. of benzene was dissolved 2.0 g. of6β-orthonitrophenylsulfenylaminopenicillanic acid parabromophenacylester. To the solution was added 60.0 g. of active manganese dioxidewith stirring at room temperature and the reaction mixture was stirredfurther for 1 hour at room temperature. After completion of thereaction, insoluble substance was removed by filtration and the solventwas removed by evaporation under reduced pressure to obtain 1.38 g. of6-orthonitrophenylsulfenyliminopenicillanic acid parabromophenacylester.

Infrared Spectrum (Nujol): 1780 cm⁻¹

Nuclear Magnetic Resonance Spectrum (CDCl₃) δppm: 1.65 (3H, singlet),1.72 (3H, singlet) 4.80 (1H, singlet), 5.43 (2H, singlet) 5.97 (1H,singlet), 7.22-8.53 (8H, multiplet)

The thus obtained 6-orthonitrophenylsulfenyliminopenicillanic acidparabromophenacyl ester was treated as in Example 4 to give6α-methoxy-6β-orthonitrophenylsulfenylaminopenicillanic acidparabromophenacyl ester.

EXAMPLE 67α-Methoxy-3-methyl-7β-paranitrophenylsulfenylamino-3-cephem-4-carboxylicacid tert-butyl ester

In 100 ml. of benzene was dissolved 2.0 g of3-methyl-7β-paranitrophenylsulfenylamino-3-cephem-4-carboxylic acidtert-butyl ester. To the solution was added 60.0 g. of active manganesedioxide with stirring at room temperature and the reaction mixture wasstirred further for 1 hour at room temperature. After completion of thereaction, insoluble substance was removed by filtration and the solventwas removed by evaporation under reduced pressure to obtain 1.65 g. of3-methyl-7-paranitrophenylsulfenylimino-3-cephem-4-carboxylic acidtert-butyl ester melting at 169°-170° C.

Infrared Spectrum (Nujol): 1790 cm⁻¹

Nuclear Magnetic Resonance Spectrum (CDCl₃) δppm: 1.58 (9H, singlet),2.15 (3H, singlet) 3.23 and 3.53 (2H, AB-quartet, J=18 Hz) 5.37 (1H,singlet), 7.55-8.40 (4H, multiplet)

In 50 ml. of anhydrous methanol was suspended 2.70 g. of thus obtained3-methyl-7-paranitrophenylsulfenylimino-3-cephem-4-carboxylic acidtert-butyl ester. To the suspension was added under stirring at -78° C.a solution of lithium methoxide which was prepared from 231 mg. ofmetallic lithium and 20 ml. of anhydrous methanol. After stirring at-78° C. for 30 minutes, 70 ml. of anhydrous tetrahydrofuran wasgradually added to the mixture and the reaction mixture was furtherstirred at -78° C. for 3.5 hours to produce a transparent solution.After 2.40 ml. of acetic acid was added to complete the reaction andthen 70 ml. of water was added to the reaction mixture, the thusobtained mixture was extracted three times with 150 ml. of ethylacetate. After washing with water and drying, the solvent was removed byevaporation under reduced pressure to obtain a crude product. The crudeproduct was submitted to purification by columnchromatography in whichsilica gel was used as a packing and a mixture of benzene-ethyl acetate(10:1) was used as an eluent to isolate 1.45 g. of7α-methoxy-3-methyl-7β-paranitrophenylsulfenylamino-3-cephem-4-carboxylicacid tert-butyl ester melting at 120°-121° C.

Infrared Spectrum (Nujol): 3300, 1765 cm⁻¹

Nuclear Magnetic Resonance Spectrum (CDCl₃) δppm: 1.52 (9H, singlet),2.12 (3H, singlet) 3.15 and 3.38 (2H, AB-quartet, J=18 Hz) 3.53 (3H,singlet), 4.48 (1H, singlet) 4.88 (1H, singlet), 7.27-8.28 (4H,multiplet)

EXAMPLE 77α-Methoxy-3-methyl-7β-paranitrophenylsulfenylamino-3-cephem-4-carboxylicacid tert-butyl ester

In 3 ml. of carbon tetrachloride was suspended 205 mg. of3-methyl-7β-paranitrophenylsulfenylamino-3-cephem-4-carboxylic acidtert-butyl ester. To the suspension was added with stirring 0.08 ml. oftriethylamine and then 73.8 mg. of N-chlorosuccinimide and the reactionmixture was stirred at 40° C. for 1 hour. After adding further 0.04 ml.of triethylamine and 42.5 mg. of N-chlorosuccinimide, the mixture wasstirred at 40° C. for 30 minutes. After completion of the reaction,insoluble substance was removed by filtration and the solvent wasremoved by evaporation under reduced pressure to obtain a crude product.The crude product was submitted to preparative silica gelthin-layer-chromatography (thickness: 0.2 cm., 20×20 cm., developmentsystem: benzene-ethyl acetate (10:1)) to isolate 134 mg. of3-methyl-7-paranitrophenylsulfenylimino-3-cephem-4-carboxylic acidtert-butyl ester.

The thus obtained compound was reacted, as in Example 5, with lithiummethoxide to give7α-methoxy-3-methyl-7β-paranitrophenylsulfenylamino-3-cephem-4-carboxylicacid tert-butyl ester.

EXAMPLE 87α-Methoxy-3-methyl-7β-orthonitrophenylsulfenylamino-3-cephem-4-carboxylicacid tert-butyl ester

(a) In 100 ml. of benzene was dissolved 2.0 g. of3-methyl-7β-orthophenylsulfenylamino-3-cephem-4-carboxylic acidtert-butyl ester. To the solution was added 60.0 g. of active manganesedioxide with stirring at room temperature and the reaction mixture wasstirred further for 1 hour at room temperature. After completion of thereaction, insoluble substance was removed by filtration and the solventwas removed by evaporation under reduced pressure to obtain 1.72 g. of3-methyl-7-orthonitrophenylsulfenylimino-3-cephem-4-carboxylic acidtert-butyl ester melting at 183°-184° C.

Infrared Spectrum (Nujol): 1780 cm⁻¹

Nuclear Magnetic Resonance Spectrum (CDCl₃) δppm: 1.60 (9H, singlet),2.16 (3H, singlet) 3.27 and 3.58 (2H, AB-quartet, J=19 Hz) 5.47 (1H,singlet), 7.33-8.62 (4H, multiplet)

(b) To a solution of 70 mg. of3-methyl-7β-orthonitrophenylsulfinylamino-3-cephem-4-carboxylic acidt-butyl ester in 1 ml. of tetrahydrofuran and 2.5 ml. of chloroform wereadded successively 93 mg. of quinoline in 0.45 ml. of chloroform and 29mg. of thionyl chloride in 0.2 ml. of chloroform. The reaction mixturewas stirred at -30° C. for 1 hour and at 0° C. for 1 hour. Further 93mg. of quinoline in 0.45 ml. of chloroform and 29 mg. of thionylchloride in 0.2 ml. of chloroform were added to the solution andstirring at 0° C. was continued overnight. To the resulting reactionmixture were added 5 ml. of saturated sodium bicarbonate solution and 50ml. of ethyl acetate, and the organic phase was separated. The aqueouslayer was extracted with 15 ml. of ethyl acetate. The combined organicextracts were dried over sodium sulfate and evaporated to give an oilwhich contained quinoline. This oil was purified by columnchromatography using silica gel to afford 36 mg. of pure3-methyl-7-ortho-nitrophenylsulfenylimino-3-cephem-4-carboxylic acidt-butyl ester, which was identical with the authentic sample obtained inthe above (a).

3-Methyl-7-orthonitrophenylsulfenylimino-3-cephem-4-carboxylic acidtert-butyl ester thus obtained was treated as in Example 1 to obtain7α-methoxy-3-methyl-7β-orthonitrophenylsulfenylamino-3-cephem-4-carboxylicacid tert-butyl ester.

EXAMPLE 97α-Methoxy-3-methyl-7β-orthonitrophenylsulfenylamino-3-cephem-4-carboxylicacid tert-butyl ester

In 15 ml. of carbon tetrachloride was suspended 656 mg. of3-methyl-7β-orthonitrophenylsulfenylamino-3-cephem-4-carboxylic acidtert-butyl ester. To the suspension was added with stirring under icecooling 0.35 ml. of triethylamine and then 585 mg. oftrichloroisocyanuric acid, and the reaction mixture was stirred at 0° C.for 1.5 hours. After completion of the reaction, insoluble substance wasfiltered off and the solvent of the filtrate was removed under reducedpressure to obtain a crude product. The crude product was submitted topreparative silica gel thin-layer chromatography (thckness: 0.2 cm.,20×20 cm., development system: benzene-ethyl acetate (10:1)) to isolate145 mg. of3-methyl-7β-orthonitrophenylsulfenylimino-3-cephem-4-carboxylic acidtert-butyl ester. The product thus isolated was treated as in Example 1to obtain7α-methoxy-3-methyl-7β-orthonitrophenylsulfenylamino-3-cephem-4-carboxylicacid tert-butyl ester.

EXAMPLE 103-Acetoxymethyl-7α-methoxy-7β-orthonitrophenylsulfenylamino-3-cephem-4-carboxylicacid benzhydryl ester

(a) In 100 ml. of benzene was dissolved 2.0 g. of3-acetoxymethyl-7β-orthonitrophenylsulfenylamino-3-cephem-4-carboxylicacid benzhydryl ester. To the solution was added 70 g. of activemanganese dioxide with stirring at room temperature and the reactionmixture was stirred for 1.0 hour at room temperature. After completionof the reaction, insoluble substance was removed by filtration and thesolvent was removed by evaporation under reduced pressure to obtain 1.40g. of3-acetoxymethyl-7-orthonitrophenylsulfenylimino-3-cephem-4-carboxylicacid benzhydryl ester melting at 134°-135° C.

Infrared Spectrum (Nujol): 1780 cm⁻¹

Nuclear Magnetic Resonance Spectrum (CDCl₃) δppm: 2.01 (3H, singlet)3.39 and 3.65 (2H, AB-quartet, J=19 Hz) 4.85 and 5.08 (2H, AB-quartet,J=14 Hz) 5.47 (1H, singlet), 7.08 (1H, singlet) 7.25-8.60 (14H,multiplet)

(b) To a solution of 122 mg. of3-acetoxymethyl-7β-ortho-nitrophenylsulfinylamino-3-cephem-4-carboxylicacid benzhydryl ester in 3 ml. of chloroform were added successively 233mg. of quinoline in 0.5 ml. of chloroform and 72 mg. of thionyl chloridein 1 ml. of chloroform under ice-water cooling. The reaction mixture wasstirred at 0° C. for 1.5 hour. To the resulting solution were added 10ml. of saturated sodium bicarbonate solution and 70 ml. of ethylacetate, and the mixture was well stirred. The organic layer wasseparated and washed with saturated sodium chloride solution twice.After drying over sodium sulfate the solvents were evaporated underreduced pressure to give an oil, which was chromatographed on 4 g. ofsilica gel. Elution with benzene and evaporation afforded 37 mg. of3-acetoxymethyl-7-ortho-nitrophenylsulfenylimino-3-cephem-4-carboxylicacid benzhydryl ester, which was identical with the authentic sampleobtained in the above (a).

3-Acetoxymethyl-7-orthonitrophenylsulfenylimino-3-cephem-4-carboxylicacid benzhydryl ester thus obtained was treated as in Example 3 to give3-acetoxymethyl-7α-methoxy-7β-orthonitrophenylsulfenylamino-3-cephem-4-carboxylicacid benzhydryl ester.

EXAMPLE 117α-Methoxy-3-methyl-7β-(2',4'-dinitrophenyl)sulfenylamino-3-cephem-4-carboxylicacid tert-butyl ester

In 100 ml. of benzene was dissolved 2.0 g. of3-methyl-7β-(2',4'-dinitrophenyl)sulfenylamino-3-cephem-4-carboxylicacid tert-butyl ester. To the solution was added 60.0 g. of activemanganese dioxide with stirring at room temperature and the reactionmixture was stirred further for 1 hour at room temperature. Aftercompletion of the reaction, insoluble substance was removed byfiltration and the solvent was removed by evaporation under reducedpressure to obtain 1.35 g. of3-methyl-7-(2',4'-dinitrophenyl)sulfenylimino-3-cephem-4-carboxylic acidtert-butyl ester melting at 187°-188° C.

Infrared Spectrum (Nujol): 1790 cm⁻¹

Nuclear Magnetic Resonance Spectrum (CDCl₃) δppm: 1.58 (9H, singlet),2.18 (3H, singlet) 3.25 and 3.60 (2H, AB-quartet, J=18.5 Hz) 5.45 (1H,singlet), 8.38-9.23 (3H, multiplet)

Thus obtained3-methyl-7β-(2',4'-dinitrophenyl)sulfenylimino-3-cephem-4-carboxylicacid tert-butyl ester was treated as in Example 2 to give7α-methoxy-3-methyl-7β-(2',4'-dinitrophenyl)sulfenylamino-3-cephem-4-carboxylicacid tert-butyl ester.

EXAMPLE 127α-Methoxy-3-methyl-7β-pentachlorophenylsulfenylamino-3-cephem-4-carboxylicacid t-butyl ester

To a solution of 500 mg. of3-methyl-7β-pentachlorophenylsulfenylamino-3-cephem-4-carboxylic acidt-butyl ester in 25 ml. of benzene was added portion wise 15 g. ofactivated manganese dioxide. The reaction mixture was stirred at roomtemperature for 1 hour. Then further 10 g. of manganese dioxide wasadded and stirring was continued for 1 hour. The solid substance wasfiltered off and washed well with benzene. The combined filtrate wasevaporated under reduced pressure and the residue was purified bypreparative tlc to afford 88 mg. of3-methyl-7-pentachlorophenylsulfenylimino-3-cephem-4-carboxylic acidt-butyl ester, which melted at 195°-197° C.

Nuclear Magnetic Resonance Spectrum (CDCl₃) δppm: 1.56 (9H, singlet),2.10 (3H, singlet) 3.22 and 3.45 (2H, AB-quartet, J=18 Hz) 5.28 (1H,singlet)

Infrared Spectrum (Nujol): 1760, 1705 cm⁻¹

To a solution of 55 mg. of3-methyl-7-pentachlorophenylsulfenylimino-3-cephem-4-carboxylic acidt-butyl ester in 3 ml. of chloroform and 2.0 ml. methanol was added asolution of 0.76 mg. of lithium in 1 ml. of methanol at -78° C. Thereaction mixture was stirred at -40° C. for 45 minutes and at -20° C.for 1.5 hours. To the resulting solution were added 8 mg. of acetic acidin 1.5 ml. of chloroform and 50 ml. of ethyl acetate, which was washedwith sodium bicarbonate solution and saturated sodium chloride solution.After drying over sodium sulfate the organic solvents were removed underdiminished pressure. The residue was purified by preparative tlc to give29 mg. of7α-methoxy-3-methyl-7β-pentachlorophenylsulfenylamino-3-cephem-4-carboxylicacid t-butyl ester as a foam.

Nuclear Magnetic Resonance Spectrum (CDCl₃) δppm: 1.49 (9H, singlet),2.08 (3H, singlet) 3.23 (3H, singlet) 3.21 and 3.31 (2H, AB-quartet,J=18 Hz) 4.79 (1H, singlet), 4.89 (1H, singlet)

EXAMPLE 137α-Methoxy-3-methyl-7β-paranitrophenylsulfenylamino-3-cephem-4-carboxylicacid tert-butyl ester

In 5 ml. of methylene chloride was dissolved 200 mg. of3-methyl-7β-paranitrophenylsulfinylamino-3-cephem-4-carboxylic acidtert-butyl ester. To the solution was added with stirring at -78° C.0.20 ml. of triethylamine and then 0.10 ml. of thionyl chloride. Afterstirring the reaction mixture at -78° C. for 1.5 hours, 20 ml. ofmethylene chloride was added thereto. After the reaction mixture waswashed with a saturated solution of sodium bicarbonate and with water,and dried, the solvent was removed by evaporation under reduced pressureto obtain a crude product. The crude product was purified by preparativesilica gel thin-layer chromatography (thickness: 0.2 cm., 20×20 cm.,development system: benzene-ethyl acetate (10:1)) to isolate 77 mg. of3-methyl-7-paranitrophenylsulfenylimino-3-cephem-4-carboxylic acidtert-butyl ester. The thus obtained compound was treated as in Example 6to give7α-methoxy-3-methyl-7β-paranitrophenylsulfenylamino-3-cephem-4-carboxylicacid tert-butyl ester.

EXAMPLE 147α-Methoxy-3-(1-methyl-1H-tetrazol-5-yl)thiomethyl-7β-ortho-nitrophenylsulfenylamino-3-cephem-4-carboxylicacid

To a suspension of 200 mg. of3-(1-methyl-1H-tetrazol-5-yl)thiomethyl-7β-ortho-nitrophenylsulfinylamino-3-cephem-4-carboxylicacid in 5 ml. of chloroform was added 44 mg. of triethylamine. After 5minutes stirring 52 mg. of chlorotrimethylsilane was added underice-water cooling and the reaction mixture was stirred at roomtemperature for 30 minutes. The resulting solution was cooled to -40° C.and 310 mg. of quinoline in 1 ml. of chloroform was added, followed bythe addition of 119 mg. of thionyl chloride. The reaction mixture wasstirred at -30° C. for 2 hours, then a solution of 36 mg. of lithium in2 ml. of methanol was added and stirring at -30° C. was continued for 2hours. The reaction was quenched by the addition of 0.3 ml. of aceticacid. Phosphate buffer (pH: 7.8, 50 ml.) was added and the aqueous layerwas separated and washed once with ether. The solution was acidified topH 3.0 with diluted hydrochloric acid and extracted with ethyl acetate.The combined extracts were washed with saturated sodium chloridesolution, dried over magnesium sulfate. Evaporation of the solvent underreduced pressure gave 200 mg. of crude7α-methoxy-3-(1-methyl-1H-tetrazol-5-yl)-thiomethyl-7β-ortho-nitrophenylsulfenylamino-3-cephem-4-carboxylicacid. For identification, this acid was converted to the correspondingbenzhydryl ester as follows. The crude acid was dissolved in 50 ml. ofethyl acetate and treated with 194 mg. of diphenyldiazomethane. Thesolution was stirred at room temperature overnight and evaporated underdiminished pressure. The residue was purified by preparative tlc usingsilica gel plate which was developed with benzene-ethyl acetate (4:1) togive 58 mg. of pure7α-methoxy-3-(1-methyl-1H-tetrazol)thiomethyl-7β-orthonitrophenylsulfenylamino-3-cephem-4-carboxylicacid benzhydryl ester as a foam.

Infrared Spectrum (Nujol): 3300, 1785, 1720 cm⁻¹

Nuclear Magnetic Resonance Spectrum (CDCl₃) δppm: 3.50 (3H, singlet),3.60 (2H, singlet), 3.76 (3H, singlet) 4.20 and 4.46 (2H, AB-quartet,J=14 Hz) 4.40 (1H, singlet), 4.92 (1H, singlet) 6.90 (1H, singlet),7.05-8.36 (14H, multiplet)

REFERENTIAL EXAMPLE 13-Methyl-7β-orthonitrophenylsulfenylamino-3-cephem-4-carboxylic acidtert-butyl ester

In 100 ml. of anhydrous tetrahydrofuran was dissolved 5.40 g. of7β-amino-3-methyl-3-cephem-4-carboxylic acid tert-butyl ester. To thesolution was added with stirring under ice cooling 2.80 ml. oftriethylamine and 40 ml. of an anhydrous tetrahydrofuran solutioncontaining 3.79 g. of orthonitrophenylsulfenyl chloride was added after30 minutes. The mixture was stirred for 2 hours under ice cooling andfurther for 3 hours at room temperature. After completion of thereaction, triethylamine hydrochloride produced was filtered off and thefiltrate was concentrated. The residue was purified bycolumnchromatography in which silica gel was used as a packing and amixture of benzene-ethyl acetate (10:1) was used as an eluent to isolate7.20 g. of3-methyl-7β-orthonitrophenylsulfenylamino-3-cephem-4-carboxylic acidtert-butyl ester melting at 171°-172° C.

Infrared Spectrum (Nujol): 3275, 1775 cm⁻¹

Nuclear Magnetic Resonance Spectrum (CDCl₃) δppm: 1.50 (9H, singlet),2.08 (3H, singlet) 3.22 and 3.60 (2H, AB-quartet, J=18 Hz) 4.73 (1H,quartet, J=5, 9 Hz) 3.67 (1H, doublet, J=9 Hz), 4.99 (1H, doublet, J=5Hz) 7.17-8.37 (4H, multiplet)

REFERENTIAL EXAMPLE 2 6β-orthonitrophenylsulfenylaminopenicillanic acidparabromophenacyl ester

In 225 ml. of anhydrous tetrahydrofuran was suspended 11.3 g. of6β-aminopenicillanic acid parabromophenacyl ester hydrochloride. To thesolution was added with stirring under ice cooling 7.0 ml. oftriethylamine, and 10 ml. of an anhydrous tetrahydrofuran solutioncontaining 4.74 g. of orthonitrophenylsulfenylchloride was added after30 minutes. The mixture was stirred for 2 hours under ice cooling andfurther for 3 hours at room temperature. After completion of thereaction, triethylamine hydrochloride product was filtered off and thefiltrate was concentrated. The residue was purified bycoloumnchromatography in which silica gel was used as a packing and amixture of benzene-ethyl acetate (10:1 V/V) was used as an eluent toisolate 17.8 g. of 6β-orthonitrophenylsulfenylaminopenicillanic acidparabromophenacyl ester.

Infrared Spectrum (Nujol): 3250, 1780 cm⁻¹

Nuclear Magnetic Resonance Spectrum (CDCl₃) δppm: 1.73 (3H, singlet),1.78 (3H, singlet) 3.67 (1H, doublet, J=10 Hz), 4.62 (1H, singlet) 4.67(1H, quartet, J=4 and 10 Hz) 5.30 and 5.55 (2H, AB-quartet, J=16 Hz)5.65 (1H, doublet, J=4 Hz), 7.18-8.42 (8H, multiplet)

REFERENTIAL EXAMPLE 33-Methyl-7β-ortho-nitrophenylsulfinylamino-3-cephem-4-carboxylic acidt-butyl ester

A solution of 590 mg. of ortho-nitrophenylsulfinyl chloride in 3 ml. oftetrahydrofuran was added to a solution of 540 mg. of7-amino-3-methyl-3-cephem-4-carboxylic acid t-butyl ester and 253 mg. oftriethyl amine in 7 ml. of tetrahydrofuran at 0° C. The reaction mixturewas stirred at 0° C. for 2 hours. The solution was diluted with 100 ml.of ethyl acetate which was washed with water, sodium bicarbonatesolution and saturated sodium chloride solution. After drying oversodium sulfate the solvents were evaporated under reduced pressure toafford an oil. This crude oil was purified by column chromatography onsilica gel to give 739 mg. of3-methyl-7β-ortho-nitrophenylsulfinylamino-3-cephem-4-carboxylic acidt-butyl ester as an oil, which is a mixture of two diastereoisomers dueto a sulfinyl group in the 7β-side chain.

Nuclear Magnetic Resonance Spectra (CDCl₃ +D₂ O) δppm: 1.46 and 1.56(9H, two singlets) 2.01 and 2.06 (3H, two singlets), 3.07 and 3.25; 3.16and 3.43 (2H, two kinds of AB-quartet, J=18 Hz), 4.34 (1H, doublet,J=4.5 Hz), 5.27 (1H, doublet, J=4.5 Hz), 7.55-8.42 (4H, multiplet)

REFERENTIAL EXAMPLE 43-(1-Methyl-1H-tetrazol-5-yl)thiomethyl-7β-orthonitrophenylsulfinylamino-3-cephem-4-carboxylicacid

In 15 ml. of methylenechloride was suspended 986 mg. of7β-amino-3-(1-methyl-1H-tetrazol-5-yl)thiomethyl-4-carboxylic acid, towhich was added 0.5 ml. of triethylamine. After 5 minutes stirring 0.5ml. of chlorotrimethylsilane was added under ice-water cooling, andstirring was continued at room temperature for 1 hour. The solution wascooled to -40° C. and 0.57 ml. of dimethylaniline and 920 mg. ofortho-nitrobenzenesulfinyl chloride in 4.5 ml. of methylene chloridewere added. The reaction mixture was stirred at 0° C. for 30 minutes andat 0° C. for 2 hours. To the resulting solution was added 50 ml. ofphosphate buffer (pH 7.8) and aqueous layer was separated, which wasonce washed with ether. The aqueous solution was acidified to pH 2.7with diluted hydrochloric acid and extracted with ethyl acetate threetimes. The combined extracts were washed with saturated sodium chloridesolution once, dried over magnesium sulfate and evaporated under reducedpressure. The residue was washed with a small amount of ether to give531 mg. of3-(1-methyl-1H-tetrazol-5-yl)thiomethyl-7β-ortho-nitrophenylsulfinylamino-3-cephem-4-carboxylicacid as powder. This compound is a mixture of two diastereoisomers dueto a sulfinyl moiety in the 7β-side chain.

Infrared Spectrum (Nujol): 3200, 1795, 1720 cm⁻¹

For identification of the each isomer, the carboxylic acids wereconverted to the corresponding benzhydryl esters by a usual manner withdiphenyl diazomethane, and isomers were separated by preparative tlcusing silica gel plate, which was developed with benzeneethyl acetate(1:1).

Nuclear Magnetic Resonance Spectrum of one isomer (DMF-d₇) δppm: 3.66(2H, singlet), 3.92 (3H, singlet), 4.25 and 4.41 (2H, AB-quartet, J=14Hz), 4.53 (1H, doublet, J=4.5 Hz), 5.50 (1H, doublet of doublet, J=4.5and 9.0 Hz), 6.98 (1H, singlet), 7.25-8.50 (14H, multiplet)

Infrared Spectrum (Nujol): 3200, 1785, 1730 cm⁻¹

Nuclear Magnetic Resonance Spectrum of another isomer (DMF-d₇) δppm:3.80 (2H, singlet), 3.95 (3H, singlet) 4.31 and 4.42 (2H, AB-quartet,J=14 Hz) 5.16 (1H, doublet, J=4.50 Hz), 5.35 (1H, doublet of doublet,J=4.50 and 9.0 Hz), 6.95 (1H, singlet) 7.25-8.51 (14H, multiplet)

Infrared Spectrum (Nujol): 3200, 1785, 1730 cm⁻¹

REFERENTIAL EXAMPLE 53-Methyl-7β-pentachlorophenylsulfenylamino-3-cephem-4-carboxylic acidt-butyl ester

To a solution of 1.35 g. of 7β-amino-3-cephem-4-carboxylic acid t-butylester and 870 mg. of propylenoxide in 20 ml. of methylene chloride wasadded dropwise a solution of 1.58 g. of pentachlorophenylsulfenylchloride in 10 ml. of methylene chloride at 0° C. After the addition thereaction mixture was stirred at room temperature for 1 hour. Thesolution was diluted with 50 ml. of methylen chloride and washed withwater three times. The dried solution (Na₂ SO₄) was evaporated underdiminished pressure to give an oil, which was chromatographed on 33 g.of silica gel. Elution with benzene and evaporation afforded 1.115 g. of3-methyl-7β-pentachlorophenylsulfenylamino-3-cephem-4-carboxylic acidt-butyl ester as a foam. Trituration with i-propyl ether gave a samplemelting at 156°-158° C.

Nuclear Magnetic Resonance Spectrum (CDCl₃ +D₂ O) δppm: 1.49 (9H,singlet, 2.03 (3H, singlet), 3.13 and 3.36 (2H, AB-quartet, J=18 Hz),4.79 (1H, doublet, J=4.5 Hz), 4.89 (1H, doublet, J=4.5 Hz)

Infrared Spectrum (Nujol): 3200, 1790, 1720 cm⁻¹

REFERENTIAL EXAMPLE 6 7β-amino-7α-methoxy-3-methyl-3-cephem-4-carboxylicacid tert-butyl ester

In a mixture of 2 ml. of methanol and 0.4 ml. of acetic acid wasdissolved 399 mg. of sodium iodide and 2 ml. of methylene chloridesolution containing 102.4 mg. of7α-methoxy-7β-paranitrophenylsulfenylamino-3-cephem-4-carboxylic acidtert-butyl ester was added to the solution under ice cooling. Aftermixture was stirred under ice cooling for 20 minutes, white crystalsprecipitated and the solution turned brown. In the solution, thestarting material was not detected by thin-layer-chromatography(development system: benzene-ethyl acetate (3:1)). Immediatelythereafter the reaction was stopped by placing the solution in a bath of-78° C. and 20 ml. of ethyl acetate was added to the solution. After thereaction mixture was quickly washed with an aqueous solution of sodiumbicarbonate, an aqueous solution of sodium thiosulfate and watersuccessively, and dried, the solvent was removed by evaporation underreduced pressure. The thus obtained residue was purified by preparativesilica gel thin-layer-chromatography (20×20 cm., thickness: 0.2 cm.,development system: benzene-ethyl acetate (3:1)) to isolate a compoundhaving a Rf value of around 0.70, thereby yielding 36 mg. of7β-amino-7α-methoxy-3-methyl-3-cephem-4-carboxylic acid tert-butylester.

Nuclear Magnetic Resonance Spectrum (CDCl₃) δppm: 1.52 (9H, singlet),2.13 (3H, singlet), 1.96-2.40 (2H, broad singlet), 3.19 (2H, singlet),3.48 (3H, singlet), 4.79 (1H, singlet)

REFERENTIAL EXAMPLE 77α-Methoxy-3-methyl-7β-phenoxyacetamido-3-cephem-4-carboxylic acidtert-butyl ester

In a mixture of 1 ml. of methylene chloride and 1 ml. of methanol wasdissolved 33.1 mg. of7α-methoxy-3-methyl-7β-paranitrophenylsulfenylamino-3-cephem-4-carboxylicacid tert-butyl ester. To the solution were added 0.1 ml. of phenylmercaptan and 0.04 ml. of triethyl amine, and the mixture was stirredfor 15 hours at room temperature. After completion of the reaction,ethyl acetate was added and the mixture was concentrated. Afterdissolving the residue in 2 ml. of methylene chloride, 0.2 ml. ofdiethylaniline was added at -40° C. and then 0.2 ml. of phenoxyacetylchloride to the solution. Thereafter the solution was stirred at -20° C.for 1 hour. After completion of the reaction, ethyl acetate was added tothe solution. The resulting solution was washed successively with anaqueous solution of potassium hydrogen sulfate, an aqueous solution ofsodium bicarbonate and water, and the solvent was removed by evaporationunder reduced pressure. The thus obtained residue was purified bypreparative silica gel thin-layer-chromatography (thickness: 0.2 cm.,7×10 cm., development system: benzene-ethyl acetate (5:1)) to isolate 27mg. of 7α-methoxy-3-methyl-7β-phenoxyacetamido-3-cephem-4-carboxylicacid tert-butyl ester. The thus yielded compound included a littleamount of the Δ² -isomer.

Nuclear Magnetic Resonance Spectrum (CDCl₃) δppm: 1.53 (9H, singlet),2.12 (3H, singlet), 3.08 and 3.33 (2H, AB-quartet, J=18 Hz), 3.55 (3H,singlet), 4.60 (2H, singlet), 5.07 (1H, singlet) 6.80-7.53 (6H,multiplet)

REFERENTIAL EXAMPLE 87α-Methoxy-3-methyl-7β-phenoxyacetamido-3-cephem-4-carboxylic acidtert-butyl ester

In a mixture of 1 ml. of methylene chloride and 1 ml. of methanol wasdissolved 30 mg. of7α-methoxy-3-methyl-7β-paranitrophenylsulfenylamino-3-cephem-4-carboxylicacid tert-butyl ester. After adding 0.1 ml. ofhexamethylphosphoroustriamide and 0.04 ml. of triethylamine, the mixturewas stirred at room temperature for 15 hours. After completion of thereaction, ethyl acetate was added and the solution was concentrated.After dissolving the residue in 2 ml. of methylene chloride, 0.2 ml. ofdiethylaniline was added at -40° C. and then was added 2 ml. ofphenoxyacetyl chloride. The resulting solution was stirred for 1 hour at-20° C. After completion of the reaction, ethyl acetate was added. Theresulting solution was successively washed with an aqueous solution ofpotassium hydrogen sulfate, an aqueous solution of sodium bicarbonateand water, and dried. The solvent was removed by evaporation underreduced pressure. The thus obtained residue was purified by preparativethin-layer-chromatography (thickness: 0.2 cm., 7×10 cm., developmentsystem: benzene-ethyl acetate (5:1)) to isolate 26 mg. of7α-methoxy-3-methyl-7β-phenoxyacetamido-3-cephem-4-carboxylic acidtert-butyl ester. This compound included a little amount of the Δ²-isomer.

REFERENTIAL EXAMPLE 97α-Methoxy-3-methyl-7β-phenoxyacetamido-3-cephem-4-carboxylic acidtert-butyl ester

After 101 mg. of7α-methoxy-3-methyl-7β-paranitrophenylsulfenylamino-3-cephem-4-carboxylicacid tert-butyl ester was dissolved in 5 ml. of acetonitrile, 0.10 ml.of phenoxyacetyl chloride was added under ice cooling and the reactionwas conducted for 30 minutes. After completion of the reaction, 1.0 ml.of ethyl acetate was added and the resulting solution was washedsuccessively with an aqueous solution of sodium bicarbonate and water.After drying the solution, the solvent was removed by evaporation underreduced pressure. The thus yielded residue was purified by preparativesilica gel thin-layer-chromatography (thickness: 0.2 cm., 10×20 cm.,development system: benzene-ethyl acetate (5:1)) to isolate 5 mg. of7α-methoxy-3-methyl-7β-phenoxyacetamido-3-cephem-4-carboxylic acidtert-butyl ester.

What is claimed is:
 1. tert-Butyl3-methyl-7-paranitrophenylsulfenylimino-3-cephem-4-carboxylate. 2.tert-Butyl3-methyl-7-orthonitrophenylsulfenylimino-3-cephem-4-carboxylate. 3.Benzhydryl3-acetoxymethyl-7-orthonitrophenylsulfenylimino-3-cephem-4-carboxylate.4. tert-Butyl 3-methyl-7-(2',4'-dinitrophenyl)sulfenylimino-3-cephem-4-carboxylate.
 5. tert-Butyl3-methyl-7-pentachlorophenylsulfenylimino-3-cephem-4-carboxylate. 6.parabromophenacyl 6-ortho-nitrophenylsulfenyliminopenicillanate.